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At least 1,207 records · Page 67Linked to original sources
PubMed2005

Evolution of the relaxin-like peptide family: from neuropeptide to reproduction.

The relaxin-like peptide family consists of relaxin-1, relaxin-2, and relaxin-3 and the insulin-like peptides (INSL)-3, INSL4, INSL5, and INSL6 (human relaxin-2 is equivalent to relaxin-1 in other species). Evolution of this family has been contentious. We therefore sought to clarify the issue by performing phylogenetic analysis of all relaxin-like peptides from the genomic databases available. Surprisingly, the phylogeny, combined with previous biologic characterizations, suggest that although relaxin's original function was likely in the brain, its reproductive role was acquired just prior to the divergence of amphibians. This phylogeny also illuminates inconsistencies in relaxin evolution in invertebrates, chickens, and cows.

Animals↗
PubMed2004

Use of the Serial Analysis of Gene Expression (SAGE) method in veterinary research: A concrete application in the study of the bovine trypanotolerance genetic control.

New postgenomic biotechnologies, such as transcriptome analyses, are now able to characterize the full complement of genes involved in the expression of specific biological functions. One of these is the Serial Analysis of Gene Expression (SAGE) technique, which consists of the construction of transcripts libraries for a quantitative analysis of the entire gene(s) expressed or inactivated at a particular step of cellular activation. Bioinformatic comparisons in the bovine genomic databases allow the identification of several up- and downregulated genes, expressed sequence tags, and unknown functional genes directly involved in the genetic control of the studied biological mechanism. We present and discuss the preliminary results in comparing the expressed genes in two total mRNA transcripts libraries obtained during an experimental Trypanosoma congolense infection in one trypanotolerant N'Dama animal cow. Knowing all the functional genes involved in the trypanotolerance control will permit validation of some results obtained with the quantitative trait locus approach, to set up specific microarrays sets for further metabolic and pharmacological studies, and to design field marker-assisted selection by introgression programs.

Animals↗
PubMed2006

Bovine transcriptome analysis by SAGE technology during an experimental Trypanosoma congolense infection.

In central and sub-Saharan Africa, trypanosomosis is a tsetse fly-transmitted disease, which is considered as the most important impediment to livestock production in the region. However, several indigenous West African taurine breeds (Bos taurus) present remarkable tolerance to the infection. This genetic capability, named trypanotolerance, results from numerous biological mechanisms most probably under multigenic dependences, among which are control of the trypanosome infection by limitation of parasitemia and control of severe anemia due to the pathogenic effects. Today, some postgenomic biotechnologies, such as transcriptome analyses, allow characterization of the full expressed genes involved in the majority of animal diseases under genetic control. One of them is serial analysis of gene expression (SAGE) technology, which consists of the construction of mRNA transcript libraries for qualitative and quantitative analysis of the entire genes expressed or inactivated at a particular step of cellular activation. We developed four different mRNA transcript libraries from white blood cells on a N'Dama trypanotolerant animal during an experimental Trypanosoma congolense (T. congolense) infection: one before experimental infection (ND0), one at the parasitemia peak (NDm), one at the minimal packed cell volume (NDa), and the last one at the end of the experiment after normalization (NDf). Bioinformatic comparisons in bovine genomic databases allowed us to obtain more than 75,000 sequences, among which are several known genes, some others are already described as expressed sequence tags (ESTs), and the last are completely new, but probably functional in trypanotolerance. The knowledge of all identified named or unnamed genes involved in trypanotolerance characteristics will allow us to use them in a field marker-assisted selections strategy and in microarrays prediction sets for bovine trypanotolerance.

Animals↗
PubMed2004

Identification and characterization of the zebrafish and fugu genes encoding tuberoinfundibular peptide 39.

Although the PTH type 2 receptor (PTH2R) has been isolated from mammals and zebrafish, only its mammalian agonist, tuberoinfundibular peptide 39 (TIP39), has been characterized thus far. To determine whether zebrafish TIP39 (zTIP39) functions similarly with the zebrafish PTHR (zPTH2R) and human PTH2Rs and to determine its tissue-specific expression, fugu (Takifugu rubripes) and zebrafish (Danio rerio) genomic databases were screened with human TIP39 (hTIP39) sequences. A single TIP39 gene was identified for each fish species, which showed significant homology to mammalian TIP39. Using standard molecular techniques, we isolated cDNA sequences encoding zTIP39. The fugu TIP39 precursor was encoded by a gene comprising at least three exons. It contained a hydrophobic signal sequence and a predicted prosequence with a dibasic cleavage site, similar to that found in mammalian TIP39 ligands. Phylogenetic analyses suggested that TIP39 forms the basal group from which PTH and PTHrP have been derived. Functionally, subtle differences in potency could be discerned between hTIP39 and zTIP39. The human PTH2R and zPTH2R were stimulated slightly better by both hTIP39 and zTIP39, whereas zTIP39 had a higher potency at a previously isolated zPTH2R splice variant. Whole-mount in situ hybridization of zebrafish revealed strong zTIP39 expression in the region of the hypothalamus and in the heart of 24- and 48-h-old embryos. Similarly, zPTH2R expression was highly expressed throughout the brain of 48- and 72-h-old embryos. Because the mammalian PTH2R was also most abundantly expressed in these tissues, the TIP39-PTH2R system may serve conserved physiological roles in mammals and fishes.

Amino Acid Sequence↗
PubMed2005

Bone morphogenetic protein-15 in the zebrafish ovary: complementary deoxyribonucleic acid cloning, genomic organization, tissue distribution, and role in oocyte maturation.

Bone morphogenetic protein-15 (BMP-15) is a member of the TGFbeta family known to regulate ovarian functions in mammals. The structure and function of BMP-15 in lower vertebrates are less known. In this study, we cloned the zebrafish BMP-15 (zfBMP-15) cDNA and depicted its genomic organization. The zfBMP-15 cDNA encodes a protein of 384 amino acids. The mature protein has 46-51% sequence identities to fugu, chicken, and mammalian BMP-15. It also shares 38-46% homology with growth and differentiation factor-9 in fishes, chicken, and mammals. Phylogenetic analysis further confirms that the zfBMP-15 is most closely related to BMP-15 from other species, whereas the growth and differentiation factor-9 peptides from fish to mammals form a distinct branch. Comparison of zfBMP-15 cDNA with zebrafish genome database revealed that zfBMP-15 is encoded by a gene with two exons and one intron, located on chromosome 6. BMP-15 mRNA is expressed in the ovary and testis and, to a lesser extent, brain, liver, gut, heart, and muscle. Real-time PCR revealed that BMP-15 is expressed in follicles at all stages of development with no significant changes over the course of folliculogenesis. Using in situ hybridization and immunocytochemistry, we detected BMP-15 in both oocytes and follicular cells. Incubation of follicles with antiserum against zfBMP15 increased oocyte maturation, whereas incubation with recombinant human BMP-15 suppressed human chorionic gonadotropin-induced oocyte maturation. These findings suggest that BMP-15 plays a role in regulating gonadal functions in fish, in particular oocyte maturation.

Amino Acid Sequence↗
PubMed2002

Cloning and distribution of galanin-like peptide mRNA in the hypothalamus and pituitary of the macaque.

Galanin-like peptide (GALP) is a newly discovered hypothalamic neuropeptide, which is regulated by leptin and implicated in the regulation of GnRH secretion in the rodent. We searched the human genome database and determined that the human GALP gene comprises six exons, as has been shown for human galanin. We used rapid amplification of cDNA ends to clone a full-length cDNA (802 bp) of the macaque homologue of GALP and found it to be highly conserved between human and macaque at both the nucleotide (93%) and peptide (94%) levels. Mature GALP is predicted to be 60 amino acids in the macaque as in other species, and the region of GALP (9-21) that shows homology to the N-terminal 13 amino acids of galanin is perfectly conserved. We mapped the distribution of GALP mRNA in the hypothalamus and pituitary of the macaque by in situ hybridization and observed that, as in rodent species, the expression of GALP mRNA is confined to the arcuate nucleus, median eminence, and neurohypophysis. Using double-label in situ hybridization, we found that nearly all (98%) GALP mRNA-expressing cells in the arcuate nucleus also express mRNA for the long form of the leptin receptor. These findings suggest that a leptin-GALP signaling pathway exists in a primate species.

Amino Acid Sequence↗
PubMed1997

Linkage of the locus for cerebral cavernous hemangiomas to human chromosome 7q in four families of Mexican-American descent.

OBJECTIVE: To determine with greater precision the map location of the locus associated with familial cavernous hemangiomas. BACKGROUND: Cavernous malformations of the brain are a significant cause of seizures, progressive or apoplectic neurologic deficit, and headache. Prevalence estimates from autopsy series vary from 0.39 to 0.9%. This disorder (OMIM #116860) can be inherited as an autosomal dominant trait with variable penetrance. Linkage to markers on the long arm of chromosome 7 was recently reported in separate reports in three apparently unrelated Hispanic kindreds as well as in two kindreds of non-Hispanic descent. DESIGN/METHODS: We examined clinically, by MRI scanning, and by pathologic examination of surgical specimens, members of four large Mexican-American families segregating cavernous hemangiomas of the brain. Linkage analysis was performed with use of blood specimens from morphologically proven cases. Two-point linkage analysis was performed with the MLINK program of the LINKAGE package. Multipoint analysis was performed between two markers and the disease locus with LINKMAP in the FASTLINKAGE package. Allele frequencies were set as described by the Genome Database (GDB). Maximum penetrance for the disease allele was set to 0.75. RESULTS: The highest lod score was observed for marker D7S652 with Zmax = 6.66 at theta(max) = 0.00. Multipoint LOD score analysis placed the disease locus in the 11 cM interval between markers D7S630 and D7S527 with Zmax = 9.19. Haplotype analysis is in agreement with the placement of the disease gene between D7S630 and D7S527 and further shows a minimal shared region within this interval, indicating a founder effect in the establishment of the mutation in these families. CONCLUSIONS: We confirmed the linkage of cavernous hemangioma to markers on the long arm of chromosome 7q, and the estimate of the map location has been refined to a region of shared haplotype between markers D7S630 and D7S527 in four Mexican-American families who may be descended from a common ancestor in Sonora County, Mexico.

Adolescent↗
PubMed2006

DIG-1, a novel giant protein, non-autonomously mediates maintenance of nervous system architecture.

Dedicated mechanisms exist to maintain the architecture of an animal's nervous system after development is completed. To date, three immunoglobulin superfamily members have been implicated in this process in the nematode Caenorhabditis elegans: the secreted two-Ig domain protein ZIG-4, the FGF receptor EGL-15 and the L1-like SAX-7 protein. These proteins provide crucial information for neuronal structures, such as axons, that allows them to maintain the precise position they acquired during development. Yet, how widespread this mechanism is throughout the nervous system, and what other types of factors underlie such a maintenance mechanism, remains poorly understood. Here, we describe a new maintenance gene, dig-1, that encodes a predicted giant secreted protein containing a large number of protein interaction domains. With 13,100 amino acids, the DIG-1 protein is the largest secreted protein identifiable in any genome database. dig-1 functions post-developmentally to maintain axons and cell bodies in place within axonal fascicles and ganglia. The failure to maintain axon and cell body position is accompanied by defects in basement membrane structure, as evidenced by electron microscopy analysis of dig-1 mutants. Expression pattern and mosaic analysis reveals that dig-1 is produced by muscles to maintain nervous system architecture, demonstrating that dig-1 functions non-autonomously to preserve the proper layout of neural structures. We propose that DIG-1 is a component of the basement membrane that mediates specific contacts between cellular surfaces and their environment through the interaction with a cell-type specific set of other maintenance factors.

Animals↗
PubMed2003

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae.

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps- phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Adenosine Triphosphatases↗
PubMed2001

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Identification, sequence and expression of a crustacean cardioactive peptide (CCAP) gene in the moth Manduca sexta.

The crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure. The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sexta central nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion (T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life, starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta.

Amino Acid Sequence↗
PubMed2007

[Characterization of novel genes regulating adipocyte differentiation].

The mid- and late stages of adipocyte differentiation are known to be regulated by transcription factors such as peroxisome proliferator-activated receptor (PPAR)gamma and CCAAT-box/enhancer binder protein (C/EBP) families. However, events in the early stage of adipocyte differentiation remain largely unknown. To gain insights into the molecular mechanisms underlying the beginning of adipocyte differentiation, we have isolated 102 genes, which are induced at the beginning of the differentiation of mouse 3T3-L1 preadipocytes, using the polymerase chain reaction (PCR)-subtraction method. Of these, 46 appear to be unknown genes. Since rapid amplification of cDNA end (RACE), cDNA library screening, and a genome database search have revealed that two of these genes are novel, we have named them factor for adipocyte differentiation (fad) 24 and fad158. The database research of amino acid sequences revealed that fad24 has a basic leucine zipper motif and an NOC domain, and fad158 has four transmembrane domains and eight leucine-rich repeats. The expression of fad24 and fad158 transiently increased after the addition of adipogenic inducers [insulin, dexamethasone, 3-isobutyl-1-methylxanthine, fetal bovine serum (FBS)]. RNAi-mediated knockdown of fad24 or antisense fad158 inhibited adipogenesis of 3T3-L1 preadipocytes and decreased expressions of PPARgamma and C/EBPalpha. Furthermore, the constitutive overexpression of fad24 or fad158 in the mouse fibroblast cell line NIH-3T3 resulted in adipocyte conversion when stimulated with adipogenic inducers and PPARgamma ligand BRL49653. Moreover, it was found that FAD24 localizes in the nucleus, especially within nuclear speckles and nucleolus, and FAD158 localizes to the endoplasmic reticulum (ER). Taken together, fad24 and fad158 appear to regulate adipocyte differentiation by activating the PPARgamma pathway.

3T3-L1 Cells↗
PubMed1997

A new hereditary cataract mouse with lens rupture.

A new cataract model originated in a recombinant inbred (RI) strain, CXS4 or CXSD (D), between BALB/cHeA(BALB/c or C) and STS/A(STS or S) mice. Opacity appeared as a white pinpoint focus in unpigmented eyes of albino mice from 5 weeks old. All the mice were bilaterally affected by 14 weeks old. They were fully viable and fertile. There was no sex difference in incidence of cataract. Histologically, the 3-4 months old mice showed vacuoles in the lens cortex. The vacuoles were spread all over the lens cortex in advanced cases. Ruptures of the lens nucleus to the vitreous chamber was a typical occurrence. For elucidation of the mode of inheritance, F1 hybrids (CXD and SXD) and backcross progenies [(CXD)F1XD and (SXD)F1XD] were analysed. No affected mice were observed in F1 hybrids. In backcross progenies, the segregation ratio of affected and normal mice was close to 1:1 in both matings. We conclude that the cataract is inherited by an autosomal single recessive gene. This mutant gene is provisionally named 'lens rupture 2' (gene symbol 1r2, Mouse Genome Database Accession No. MGD-JNUM-37399). The new cataract model mouse will be a good tool for the genetic analysis and molecular biological study of cataractogenesis.

Age Factors↗
PubMed2006

Characterization of Sulfolobus solfataricus 2-keto-3-deoxy-D-gluconate kinase in the modified Entner-Doudoroff pathway.

The thermoacidophilic archaeon Sulfolobus solfataricus is known to utilize D-glucose via the nonphosphorylated Entner-Doudoroff (ED) pathway. But, the genome database shows that this microorganism has a gene (kdgK) encoding 2-keto-3-deoxy-D-gluconate (KDG) kinase (KDGK) which phosphorylates KDG to 2-keto-3-deoxy-6-phosphogluconate. Interestingly, kdgK and three other genes in the modified ED pathway are organized as an operon-like structure. In this study, we report confirmation of the catalytic activity of the S. solfataricus KDGK protein. We also found that the kdgK gene was transcribed as polycistronic transcripts. Proteome analysis of cell lysate revealed that all gene products in the kdgK operon were expressed as functional proteins. These results strongly indicate that S. solfataricus metabolizes D-glucose via the 'partially' nonphosphorylated ED pathway. A purified recombinant S. solfataricus KDGK had K(m) and k(cat) values of 0.14 mM and 60.8 s(-1) respectively for KDG, and showed maximal activity at temperatures between 70 and 80 degrees C and pHs between 7.0 and 8.0.

Amino Acid Sequence↗
PubMed1996

A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli.

In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affects the EnvZ function are discussed.

Amino Acid Sequence↗
PubMed2006

Analysis of the hyperthermophilic chitinase from Pyrococcus furiosus: activity toward crystalline chitin.

Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the beta-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.

Amino Acid Sequence↗
PubMed2001

A novel membrane protein complex on the endoplasmic reticulum and early Golgi compartments in the yeast Saccharomyces cerevisiae.

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therefore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.

Amino Acid Sequence↗
PubMed2005

New deblocking aminopeptidases from Pyrococcus horikoshii.

It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 degrees C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.

Amino Acid Sequence↗